Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats, obtained from healthy donors, by separation on Biocoll Separating Solution (Biochrom, L6115), according to standard procedures. Non-adherent cells were separated from PBMCs after a 30 min adherence step at 37 °C. Next, CD4 + T cells were purified from remaining non-adherent cells using the Human Resting CD4 + T cell Isolation Kit (STEMCELL Technologies, 17962) and following manufacturer´s instructions. For T cell stimulation, we treated CD4 + T cells with either αCD3αCD28 (ImmunoCult™ Human CD3/CD28 T Cell Activator; STEMCELL Technologies, 10971) or IFN I (1:1000, Human IFN Alpha Hybrid (Universal Type I IFN); PBL ASSAY SCIENCE, 11200-1). Cells were cultured in RPMI 1640 (Gibco), supplemented with 10% fetal bovine serum (FBS, Sigma), 20 mM Hepes (Hyclone), 0.3 mg/mL L-glutamine (Hyclone), 100 U/mL penicillin (Gibco) and 100 μg/mL streptomycin (Gibco).
For nucleofection experiments, total human CD4 + T cells were isolated with EasySep Human CD4 + T Cell Isolation Kit (STEMCELL Technologies, 17,952) and cultured in X-VIVO 15 (Lonza). Cells were stimulated with αCD3αCD28 (ImmunoCult™ Human CD3/CD28 T Cell Activator; STEMCELL Technologies, 10971).
For nucleofection experiments, total human CD4 + T cells were isolated with EasySep Human CD4 + T Cell Isolation Kit (STEMCELL Technologies, 17,952) and cultured in X-VIVO 15 (Lonza). Cells were stimulated with αCD3αCD28 (ImmunoCult™ Human CD3/CD28 T Cell Activator; STEMCELL Technologies, 10971).